Making l-glutamic acid alone or a mixture of l-glutamic acid and alpha-ketoglutaric acid



v used methods in this art.

United States Patent Patented Feb. 2%), 1962 MAKING L-GLUTAMIC AClD ALONE OR A MEX- TURE 0F L-GLUTAMZC ACID AND a-KETO- GLUTARIC ACID Eric A. Bore], Wilmington, Del, assigncr to Hercnies Powder Company, Wilmington, Del., a corporation oi Deiaware No Drawing. Filed June 10, 19nd, Ser. No. 35,124

6 Claims. (Cl. 195-29) The present invention relates to a process of producing a mixture of u-ketoglutaric acid and L-glutamic acid or L-glutamic acid alone and more particularly to microbiologically converting D-glutamic acid in a racemic mixture of DL-glutamic acid to a mixture of a-ketoglutaric acid and L-glutamic acid or to L-glutamic acid alone without substantially atfecting the L-glutarnic acid in the racemic mixture.

My copending application, Serial No. 35,118, entitled Biosynthesis of a-Ketoglutaric Acid, filed on even date herewith, includes a process of biologically converting D- glutamic acid in a raccmic mixture of DL-glutamic acid to u-ketoglutaric acid without substantially affecting the L-glutamic acid, using a microorganism of the genus Aerobacter. It is pointed out in said copending application mat all known methods of making L-glutamic acid by chemical synthesis result in his 50/50 or racemic mixture of L-glutamic acid and D-glutamic acid, that the mixture is useless as such, that the two acids are extremely difficult to separate in the mixture, and that only the L- glutamic acid by itself is the useful acid.

While the process of said copending application gives very good yields, these yields could be increased still further if one could reduce or eliminate the microorganisms consumption of a-ketoglutaric acid. (The microorganism consumes some of the a-ketoglutaric acid product and some of the D-glutarnic acid to grow on.) In attempting to do this I isolated mutants of the microorganism Aerobacter and used them instead of the wild type Aerobacter.

Such mutants were isolated following irradiation of the Wild type strain with ultraviolet light. After irradiation to a point Where approximately 99.5% of the microorganisms were killed, the surviving microorganisms were cultivated in the presence of succinate. The resulting population Was then screened for mutants giving a growth response to succinate. The isolation or screening was by one of a number of equivalent well-known and widely From the data given in the examples hereinafter, it is apparent that such mutants were impaired in the conversion of oe-ketoglutaric acid to suctrypsin. This tryptic digest is commercially available un-.

der the designation NZ -Case. I have found that a number of these mutants give the surprising results in accordance with this invention.

To my surprise I found that the above mutants not only consumed less u-ketoglutaric acid than the wild type from which there were produced but the mutants also produced substantial amounts of L-glutamic acid in addition to a-ketoglutaric acid, and one of the mutants produced a substantial amount of L-glutamic acid alone. This is a decided advantage because usually L-glutamic acid is the more desired acid and often where wketoglutaric acid is the acid produced, one will convert it to L-glutamic acid either by an additional microbiological process or by chemical means. Since the conversion from u-ketoglutaric acid to L-glutamic acid is not etficient, the desirability of going directly to L-glutamic acid instead of via a-ketoglutaric acid is obvious.

I have found that these desirable and surprising results are obtained by carrying out the process which comprises cultivating under aerobic conditions in a nutrient medium including D-glutamic acid, a mutant having the enzyme D-glutamic acid oxidase, the mutant having been obtained from a microorganism of the genus Aerobacter having the enzyme D-glutamic acid oxidase, said cultivation being carried out until a substantial quantity of the D-glutamic acid is converted to a mixture of a-ketoglutaric acid and L-glutamic acid or to L-glutamic acid alone.

The D-glutamic acid in the nutrient medium may be the compound D-glutamic acid as such or it may be D- glutamic acid in admixture with L-glutamic acid either as a racemic mixture, i.e. equal proportions of D-glutamic acid and L-glutarnic acid, or a mixture of D-glutamic acid and L-glutamic acid in any other proportions; the recitation in the claims including D-glutamic acid is intended to include either of these or any combination thereof. However, one is more apt to be using the racemic mixture.

The following examples illustrate various specific embodiments of this invention, but they are not intended to limit the invention beyond the scope of the claims of this application. In the examples and elsewhere herein percent and parts are by weight. The L-glutamic acid, D- glutamic acid, and the a-ketoglutaric acid were determined by well-known analytical methods commonly used in this art. The L-glutamic acid was determined by the Warburg manometric method used by the Glutamate Manufacturing Association (Methods in Enzymology by Colowick and Katlan, vol. 2, 1955, p. 182, New York Academic Press). The D-glutamic acid and the a-ketoglutaric acid were determined by paper chromatography. The a-ketoglutaric acid was also determined by the Friedemann and I-Iaugen colorimetric method (Friedemann, T. E., and Haugen, G. E., J. Biol. Chem. 147, 415, (1943) For the sake of brevity, the following abbreviations are used in the examples of this application: DL-ga means DL-glutamic acid. D-ga means D-glutamic acid. L-ga means L-glutamic acid and a-kga means a-ketoglutaric acid. 7

I have deposited the mutants of this invention with the American Type Culture Collection (ATCC), and they have given them the ATCC numbers indicated hereinafter.

In the examples 100 ml. portions of an aqueous growth medium of about neutral pH containing 0.5 g. D-glutamic acid and salts in 500 ml. Erlenmeyer flasks were inoculated with 0.1 ml. portions of the mutants liquid stock cultures and incubated at 30 C. in a rotatory shaker. (The stock cultures were considered to contain approxi:

. 3 mately microorganisms per M 550 K HPO and NaCl. The results obtained are given in Tables 1 and 2 hereinafter.

Surprisingly, none of the mutants used in the following Table 1 SUCCINATE MUTANTS ml.) The salts were pH outside this range is applicable but the conversion rate is slower.

Only those mutants having the enzyme D-glutamic acid oxidase and obtained from a microorganism of the genus Aerobacter having the enzyme D-glutamic acid oxidase are operable in this invention. Alarge number of Aerobacter mutants not having said enzyme were tested under e conditions of this invention and theydid not produce any L-glutamic acid or wketoglutaric acid. V

This invention has been describedwith'reference to specific embodiments thereof in which a growth medium is inoculated with a microorganism; and without any special treatment or modification of the microorganism, it efiects the desired conversion while growing on the medi- Y 1 um. Thus it is not necessary to become involved with Example N0 1 2 3 making cell-free preparations or resting cell preparations ATCOV from the microorganism and then using these prepara- 1mubitio%arae;'n;::::3:::::::::::::iii. it? tions to effect the d n i Hvwcver, reams eg Converswn t a sed on N 9 cell or cell-free preparations, or isolated pure enzymes,

A Percent Conversion to lrga based on 3 r 20 can be used within the scope of th s invention. igg f 30 20 7 Thus the present 1nvent1on provides an effective means amount 0? L-ga m 100 1G0 1% whereby the D-glu'tamic acid isomer in a DL-glutamic Dga Y 110 acid racemate may be converted to: either L-glutamlc acid alone or to a mixture of L-glutamic acid and a-ketoas ExamplellfTrable glutaric acid without substantially atfecting the L-glu- Table 2 NZ-CASE MUTANTS Percent Con- Percent L-ga Growth Optiversion to as Compared Percent Yield cal Density pH Change wkga Based to Initial Based on D-ga Left at 650 my. on D-ga Amlount oi D-ga 7 Example No. A 'lIOC Incubation Time, hrs. Incubation a-kga Time, hrs.

and L-ga L-ga 24 48 24 48 24 48 24 48 24 48 129 1.0 1.10 8.3 8.9 39.6 44.4 100 90 32c None No No. 13814 .17 .55 7.0 8.5 4.4 36 100 115 51.0 15 Yes... 'No. 13815 46 .80 7.8 8.7 29.4 37.8 112 100 41.4 12 Yes. No. 13816 38 1.00 7 8 8.8 33.6 43.2 112 80 45.6 12 Yes--- No. 13817 51 .88 7.8 8.7 33.6 37.8 121 100 54.6 21 No No. 13818 49 .78 7.9 8.7 43.2 42.6 114 100 57.2 14 No- No. 13819 52 .80 7.8 8.7 84.2 48 110 100 48.0 10 No No. 13820 42 .79 7.8 8.7 36 48 112 80 48.0 a 12 Yes... No.

Example 1 Control. Aerobacter acrog enes ATCC 129 from which mutants of Examples 5-11 were prepared.

than didthe control which was the parental wild type from which the mutants were obtained (Example 1).

As those skilled in this art will appreciate, the conditions set forth in the above examples may be varied contamic acid isomer of the racemate. As disclosed more fully hereinbefore, the yields are unexpectedly high. The L-glutamic acid and u-ketoglutaric acid can be separated easily by known means. The a-ketoglutaric acid can be used as such or more L-glutamic acid can be synthesized from it by either chemical or microbiological means well j known in this art.

siderably within the scope of the present invention defined undesirable to operate below about 25 C. Generally,

there is no appreciable advantage in operating above about C. The microorganisms are apt to be seriously damaged, if not killed, at a temperature of about C.

, ApH of 6-9 is satisfactory as the initial pH of the growth medium, but a pH of about 7.5 is preferred. A

As many apparent and widely difierent embodiments of this invention may be made without departing from the spirit and scope thereof, it is to be understood that the invention is not limited to the specific embodiments thereof except as defined in theappended claims.

1. Process of preparing L-glutamic acid comprising cultivating under aerobic conditions in a nutrient me-' dium including D-glutamic acid, a mutant selected from the group consisting (a) of those characterized by impairment in the conversion of a-ketoglutaric acid to sucj cinate and (b) of those characterized by giving a growth response to hydrolyzed casein, said mutant being obtained from a microorganism of the genus Aerobacter and both the mutant and parential wild type from which the mutant is obtained havingthe enzyme D-glutamic acid oxidase, said cultivation being carried out until a substantial quantity of the D-glutamic acid is converted to L-glutamic acid. V

2. Process of claim 1 wherein the mutant employed What I claimand desire to protect by Letters Patent I is characterized by impairment in the conversion of uketoglutaric acid to succinate.

3. Process of claim 1 wherein the mutant employed is characterized by giving a growth response to hydrolyzed casein.

4. Process of claim 1 wherein the mutant employed is obtained from a microorganism of the genus Aerobacter and species aerogenes.

5. Process of claim 1 wherein the mutant employed is obtained from Aerobacter aerogenes ATCC 129.

6. Process of claim 1 wherein the cultivation is carried out until a substantial quantity of D-glutamic acid is converted to a mixture of L-glutamic acid and wketoglw taric acid and the latter two acids are separated.

References Cited in the file of this patent Izaki et al.: Bull. Agr. Chem. Soc, Japan, volume 19, pages 233-239 (1955), and volume 22, pages 78-84, abstracted in Chemical Abstracts, volume 50, 13l66F (1955) and volume 52, 18662b (1958), respectively.

Otsuka et al.: Chemical Abstracts, volume 51 1957), 764311 to 7644c. 

1. PROCESS OF PREPARING L-GLUTAMIC ACID COMPRISING CULTIVATING UNDER AEROBIC CONDITIONS IN A NUTRIENT MEDIUM INCLUDING D-GLUTAMIC ACID, A MUTANT SELECTED FROM THE GROUP CONSISTING (A) OF THOSE CHARACTERIZED BY IMPAIRMENT IN THE CONVERSION OF A-KETOGLUTARIC ACID TO SUCCINATE AND (B) OF THOSE CHARACTERIZED BY GIVING A GROWTH RESPONSE TO HYDROLYZED CASEIN, SAID MUTANT BEING OBTAINED FROM A MICROORGANISM OF THE GENUS AEROBACTER AND BOTH THE MUTANT AND PARENTIAL WILD TYPE FROM WHICH THE MUTANT IS OBTAINED HAVING THE ENZYME D-GLUTAMIC ACID OXIDASE, SAID CULTIVATION BEING CARRIED OUT UNTIL A SUBSTANTIAL QUANTITY OF THE D-GLUTAMIC ACID IS CONVERTED TO L-GLUTAMIC ACID. 